A measured step in our understanding of helicases
University of Illinois researchers tie structure of helicase
molecule to its step size as it unzips and re-zips the DNA strand.
The work has implications for the study of genetic disorders and
genetic susceptibility to disease.
for Illinois Physics
A team of physicists and chemists at Illinois are uncovering exactly how enzymes take apart the DNA double helix and then put it back together. A recent study combining computational and theoretical biophysics with advanced experimental methods offers an atomically detailed picture of how enzymes called helicases move along a DNA molecule as it is unzipping its strands or re-zipping them. These findings were published in the journal Nature Communications in the article “Kinetic and structural mechanism for DNA unwinding by a non-hexameric helicase.”
Helicases are a class of motor proteins, enzymes that use energy from the chemical reaction they catalyze to move along the backbone of molecules such as DNA. Many helicases play important roles in fundamental biological processes such as DNA replication, where they unwind the two DNA strands before each is copied. The new study focused on a type of helicase called UvrD, which is typically involved in DNA repair pathways in E. coli, explains Sean Carney, the article’s first author and a doctoral student in Illinois Physics Professor Yann Chemla’s research group. DNA can be damaged during some common cellular processes or by environmental factors such as uv light. Since damage to DNA can be lethal, all cells are equipped with machinery to repair their genome.
Detailing that process at the molecular level can consequently have implications for human health. Carney explains, “Helicases are linked to cancer, genetic disorders, genetic predisposition to cancer, and premature aging.”
He and collaborators studied a UvrD helicase while it moved along a piece of DNA shaped like a hairpin and either took its strands apart, unzipping them, or re-zipped them together. Helicases move along the DNA backbone in a stepwise manner, much like a climber may move along a rope ladder.
One of the big insights of the new study is pinpointing the step size for the enzyme or, in the analogy, the separation between the rungs that the climber steps on moving up or down the ladder. The Illinois team experimentally determined that during both unzipping and zipping of the DNA, UvrD helicase moved three base pairs at a time. Additionally, the team conducted sophisticated molecular dynamics simulations in order to understand structural reasons behind this step size, revealing that UvrD sequesters single DNA strands in loops before periodically releasing them.
“Our general goal was to understand the mechanism of DNA unwinding and specifically to quantify the step sizes for each discrete cycle of motion of the helicase along the DNA track. How many base pairs is the helicase unwinding at a time? How long does it dwell between steps?” Carney expands.
To determine these details experimentally, Illinois researchers used an experimental tool called optical tweezers. Here, each end of the DNA hairpin was connected to a polystyrene bead, which was in turn held by a beam of carefully focused laser light. When the helicase moved along the DNA and separated its strands (in the analogy, the climber undoing the ladder while moving upwards), one of the beads would move as well. In particular, the experiment was set up so that the tension of the DNA suspended between the beads had to be maintained at all times. When the helix got unwound and elongated, the connection between the beads went slack, and to become taut again, one of the beads would have to move. By recording the motion of this bead, researchers could determine how much longer the DNA had become. They then converted this information into how many base pairs UvrD had stepped over and unwound.
Carney emphasizes, “We can directly measure steps as small as an individual base pair. We can look at sub-nanometer-level stepping dynamics in high resolution. Similar measurements have been done before for other helicases, but this is the first work to directly measure the step size with base pair resolution specifically for the UvrD helicase. Because UvrD shares the same unwinding mechanism with other unrelated helicases, this is of great interest to the biophysics and chemistry research community.”
He and collaborators, however, did not stop there. They complemented their precise experimental work with computer simulations performed by theorists in the research group of Illinois Chemistry and Physics Professor Zaida Luthey-Schulten.
Wen Ma, at the time of this experiment a doctoral student in Luthey-Schulten’s group and currently a postdoctoral researcher at University of California San Diego, explains, “We simulated the UvrD-DNA complex at the atomistic level. The motions of hundreds of thousands of atoms were computed using Newton’s laws. We saw how the single-stranded DNA tails formed from the unwound helix’s interacting with UvrD.”
Ma’s simulations showed that the enzyme sequesters single strands of DNA into loops as it moves along the strand, and it takes the single strands apart, instead of letting them go immediately after unzipping. When UvrD releases three base pairs, the DNA loop becomes extended, causing the slack that made the polystyrene beads in Carney’s experiment move. The predicted step size distribution from simulations agrees well with the experimental distribution. “Our simulations show that the atomistic-level details are important in understanding the step size distributions measured in single-molecule experiments,” Ma asserts.
Carney says, “Including this theoretical and computational component in our work further illuminates what we are observing. The agreement between theory and experiment regarding the UvrD step size validates our observations, and the simulations provide additional insight into the mechanism of unwinding.”
Further, these simulations identify the specific part of the UvrD protein that contacts the DNA. While the delayed release mechanism has been proposed before, such a precise understanding of what part of the protein is responsible for loop formation was never before available to researchers.
“Additionally, previous work in the field only proposed models for DNA unwinding. Here, as a new addition, we also measure the stepping dynamics of DNA zipping and propose a mechanism for the zipping process similar to that for unwinding,” Carney adds.
Going forward, the team wants to test its theory by using a mutated UvrD molecule that they predict will, as a consequence of mutation, move along the DNA with a different step size. Carney explains that detecting such a change in step size based on structural properties of the UvrD would offer the additional empirical confirmation to experimental and computational work researchers have done already.
“It would also be very interesting to show that the mechanism proposed for how exactly UvrD unzips and rezips the DNA can be applied more generally than to just this one helicase enzyme,” Ma says. He is eager to broaden the impact of the work. “We found the loop-forming protein sites are consistent across a group of structurally similar helicases, called superfamily I helicases, based on bioinformatics analysis. We think that the mechanism might be utilized by other helicases in this superfamily.”
This research was funded by the National Institutes of Health under Grant Nos. R01 GM120353, R01 GM45948, and R35 GM136632; and by the National Science Foundation Physics Frontiers Center (PFC) program under Grant No. PHY-1430124 (Center for the Physics of Living Cells). The findings presented are those of the researchers, and not necessarily those of the funding agencies.